INFLUENCE OF ALPA-HELICES ON THE FOLDING OF APAMIN

ALPA-螺旋对 APAMIN 折叠的影响

• 批准号: 3466879
• 负责人: JEFFREY W NELSON
• 金额: $ 9.81万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3466879
• 关键词: chemical bond; chemical structure; circular dichroism; disulfide bond; insect poison; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein engineering; protein folding; protein sequence

The aim of this research is to understand how the stability of an alpha-helix influences the pathway and kinetics of protein folding. Apamin, a small bee venom protein which forms an alpha-helix on the C-terminal end and contains two disulfide bonds, will be used as the model system. The mechanism for folding from the unfolded state of apamin, with reduced thiols, to the native folded state, with two disulfide bonds, will be studied by trapping the disulfide intermediates with iodoacetic acid throughout the folding process. Separation of the intermediates by HPLC will determine the rates acid throughout the folding processes. Separation of the intermediates by HPLC will determine the rates of build-up and decay of each intermediate, while enzymatic cleavage, followed by separation of the fragments, will tell which cysteines are linked by disulfide bonds. In order to study the stability of the alpha-helix in apamin, the C- terminal helical part of apamin will be synthesized by solid phase peptide methods, replacing the cysteines by alanines to avoid disulfide bond formation. The stability will be studied by using the helix-stabilizing properties of trifluoroethanol. Alpha-Helix formation will be studied by circular dichroism, to measure overall helicity, and by two-dimensional NMR, to monitor the helical transition of each amino acid. After understanding the folding of natural apamin and the stability of its helix, apamin derivatives with substitutions on the C-terminal helix will be synthesized. Comparisons of the folding kinetics and helical stability of the derivatives with natural apamin will provide information about the relationship between alpha-helix stability and folding kinetics. This research will provide new insight into how the stability of an alpha-helix influences the folding of small proteins.

本研究的目的是了解稳定性如何 α-螺旋影响蛋白质折叠的途径和动力学。 Apamin，一种小型蜂毒蛋白，在其上形成 α 螺旋 C 末端并含有两个二硫键，将被使用 作为模型系统。 折叠机构 apamin 的未折叠状态，具有还原的硫醇，至天然折叠状态 具有两个二硫键的状态，将通过捕获来研究 与碘乙酸的二硫化物中间体 折叠过程。 通过 HPLC 分离中间体 确定整个折叠过程中的酸速率。 通过 HPLC 分离中间体将确定速率 每个中间体的积累和衰变，同时酶促 裂解，然后分离片段，将告诉您哪个 半胱氨酸通过二硫键连接。 为了研究 apamin 中 α 螺旋的稳定性，C- apamin 的末端螺旋部分将通过固相合成 肽方法，用丙氨酸替换半胱氨酸以避免 二硫键的形成。 稳定性将通过使用来研究 三氟乙醇的螺旋稳定特性。 阿尔法螺旋 将通过圆二色性研究形成，以测量 整体螺旋度，并通过二维 NMR 来监测 每个氨基酸的螺旋转变。 了解天然 apamin 的折叠和 其螺旋的稳定性，apamin 衍生物的取代 将合成C-末端螺旋。 折叠方式比较 天然衍生物的动力学和螺旋稳定性 apamin 将提供有关之间关系的信息 α-螺旋稳定性和折叠动力学。 这项研究将 提供关于α螺旋稳定性如何的新见解 影响小蛋白质的折叠。